The overall goal of this work is to develop anatomical, functional, and molecular magnetic resonance imaging (MRI) techniques that allow non-invasive assessment of brain function and apply these tools to study plasticity and learning in the rodent brain. MRI techniques are having a broad impact on understanding brain. Anatomical based MRI has been very useful for separating gray and white matter and detecting numerous brain disorders. Functional MRI techniques enable detection of regions of the brain that are active during a task. Molecular MRI is an emerging area, whose major goal is to image a large variety of processes in tissues. The goal of this project is to translate MRI developments in all these areas to study system level changes that occur in the rodent brain during plasticity and learning. Aim 1: Over the past few years, we have completed studies in the rodent brain that acquired very high temporal and spatial resolution functional MRI (fMRI) to monitor changes in hemodynamics as a surrogate marker of electrical activity during forepaw stimulation. Work over the past year has been completed that has acquired very high signal to noise fMRI maps to determine if the borders of activated areas can be well defined. The borders determined were used to study plasticity of cortical representations after peripheral nerve damage. Over the past year we have demonstrated that these high resolution rodent fMRI techniques can map the expansion of the cortical nose representation into the whisker barrel region afer damage to whiskers in young animals. The nose region grew selectively as compared ot the forelimb or hindlimb. These studies also demonstrated that multiple regions of the rodent cortex could be mapped in individuals. Aim 2: Over the past several years we have demonstrated that manganese chloride enables MRI contrast that defines neural architecture, can monitor activity, and can be used to trace neural connections. Over the last couple of years we have completed the assignment of cortical layers detected using manganese enhanced MRI by comparison to histology and have demonstrated that functional anatomy of several cortical regions of the rodent brain can be defined in individual animals. In particular, clear cytoarchitectural boundaries can be detected between numerous brain areas enabling, for the first time, cytoarchitectural changes to be followed in individual brains over time. We have also demonstrated that activity in the olfactory bulb can be imaged to the level of single glomeruli using manganese enhanced MRI and we have obtained evidence that indicates the flow of neural information from the glomerular to mitral layer can be imaged. Over the past year we have confirmed that manganese based track tracing can image connections to the level of specific layers. This has been used to trace the laminar inputs of the olfactory pathway from the olfactory bulb to rodent frontal cortex. The anatomic projections from olfactory cortex to frontl cortex have not previously been measured. The manganese based MRI predictions are being confirmed by classical histological based neural tracing techniques. Aim 3: Functional MRI studies were performed to measure changes in brain activation that occur after denervation of peripheral nerves. After severing the saphanous and sciatic nerve of one hindpaw, the good hindpaw is now able to cause activation of about 50% of the damaged hindpaw's cortical representation even though it is in the opposite brain hemisphere. Lesion experiments support the model that cortical-cortical communication via the corpus collosum is responsible for this plasticity. Similar results were obtained after severing the nerves that innervate the forepaw and looking at fMRI activation of the good forepaw. High resolution fMRI indicates that the activation in the damaged cortex is about 30% of the normal amplitude and occupies about 50% of the representation. Additionally the good cortex activation increases by a factor of about two. to understand the neural basis of these changes electrophysiology was performed. Consistent with the increased fMRI in the good cortex there was an increase in local field potentials and an increase in the number of single units that responded to stimulation from about 30% of cells to 60% of cells. Interestingly in the cortex ipsalateral to the good paw, no significant local field potential could be detected even though signifcant fMRI was detected. To the best of our knowledge this is the first time fMRI and local field potentials do not correlate without using pharmacological manipulation. Single unit recordings found a number of cells that responded to stimulation on the ipsalateral side. The majority of cells had short action potential duration and juxtapositional labeling indicates that these cells are interneurons. Thus, the fMRI activation is attributed to increased interneuron activity without significant pyramidal cell activiation in this model of injury induced plasticity. This represents the first time in the intact brain where fMRI has been caused by interneuron acitivity. This has far reaching implications for the analysis of fMRI data. Furthermore, the fact that the ipsalateral cortex is in an increased state of inhibition has behavioral consequences which will be explored in the coming year. Similar fMRI results have been obtained on the whisker barrel cortex over the past year. Finally, manganese based MRI track tracing indicates changes in innervation in later 4 in the good cortex and layer 3 in the ipsalateral cortex have occurred. These results will be used to guide elecrtophysiology experiments in slice to determine the synaptic changes that occur in this model. Aim 4: We have begun to explore the use of advanced MRI tools for studying simple learning paradigms in the rodent. In order to accomplish this we have been developing techniques that will enable routine fMRI in awake rodents. While fMRI is widely performed in humans and awake primates there have only been a few scattered studies on awake rodents. Training regimens and techniques to hold the head have been developed and intial fMRI results are being obtained. The awake rodent fMRI protocl will first we used to study brain changes during olfactory induced fear conditioning.